Pseudomonas aeruginosa is an opportunistic pathogen of humans commonly isolated from patients with cystic fibrosis, nosocomial pneumonias, urinary tract infections, or severe burns. One of the many virulence determinants of P. aeruginosa is a type III secretion system. The type III system is required for pathogenesis in infection models of lung, cornea, and burned epithelia and functions to deliver the ExoS, ExoT, ExoU, and ExoY cytotoxins to the cytoplasm of eukaryotic host cells. Translocation of these toxins promotes evasion of host immune responses and dissemination of P. aeruginosa from sites of colonization. Expression of the type III system is highly regulated and induced through contact of P. aeruqinosa with eukaryotic cells, growth in the presence of serum, or tow Ca2+ concentrations. The mechanism of coupling these environmental cues to expression of the regulon are unclear. The long-term goal of these studies is to identify and characterize the mechanisms involved in regulation of the P. aeruginosa type III regulon. We have identified ExsD as a negative regulator of the type III regulon. ExsD is unique to P. aeruginosa, lacks a DNA-binding motif, and is coordinately regulated with the type III regulon. In an exsD mutant expression of the regulon is derepressed and overexpression of ExsD leads to repression of the regulon. We hypothesize that ExsD functions as a sensor of the type III translocase. Prior to contact of P. aeruginosa with eukaryotic host cells the type Ill secretion channel is closed and ExsD prevents expression of the type III system. Contact with host cells opens the type III translocase and triggers a mechanism for suppressing the negative regulatory activity of ExsD. Therefore, we hypothesize that the negative regulatory activity of ExsD is reflective of the secretion state of the bacterial cell. This would provide a simple and specific mechanism for induction of the type III system in response to contact of P. aeruginosa with eukaryotic cells. To test these hypotheses we propose the following specific aims; (1) Identify ExsD domains and residues important for negative regulation, (2) Identify and characterize ExsD interaction partners, and (3) Determine the regulatory role of ExsD in response to alternative environmental cues. [unreadable] [unreadable]